Dendritic effects of genetically encoded actin-labeling probes in cultured hippocampal neurons

Abstract

Actin cytoskeleton predominantly regulates the formation and maintenance of synapses by controlling dendritic spine morphology and motility. To visualize actin dynamics, actin molecules can be labelled by genetically fusing fluorescent proteins to actin monomers, actin-binding proteins or single-chain anti-actin antibodies. In the present study, we compared the dendritic effect of EGFP-actin, LifeAct-TagGFP2 (LifeAct-GFP) and Actin-Chromobody-TagGFP2 (AC-GFP) in mouse cultured hippocampal neurons using unbiased quantitative methods. The actin-binding probes LifeAct-GFP and AC-GFP showed similar affinity to F-actin, but in contrast to EGFP-actin, they did not reveal subtle changes in actin remodelling between mushroom shaped spines and filopodia. All tested actin probes colocalized with phalloidin similarly, however, the enrichment of LifeAct-GFP in dendritic spines was remarkably lower compared to the other constructs. LifeAct-GFP expression was tolerated at a higher expression level compared to EGFP-actin and AC-GFP with only subtle differences identified in dendritic spine morphology and protrusion density. While EGFP-actin and LifeAct-GFP expression did not alter dendritic arborization, AC-GFP expressing neurons displayed a reduced dendritic tree. Thus, although all tested actin probes may be suitable for actin imaging studies, certain limitations should be considered before performing experiments with a particular actin labelling probe in primary neurons. [Media: see text] [Media: see text]

A methodology for specific disruption of microtubule polymerization into dendritic spines

“…These results suggest that the plasmid constructs are localizing as expected in mature hippocampal neurons following short-term expression. Although a recent publication indicated LifeAct was less concentrated in spines than other actin labels ( Ignacz et al. , 2023 ), we detected very low levels of LifeAct label in the dendrite shaft and significant enrichment in dendritic spines.…”